Effects of substrate stiffness on bacterial biofilm formation

Biofilms are communities of microbial cells attached on surfaces and embedded in a self-produced extracellular matrix comprised of polysaccharides, DNA, and proteins. Biofilms of pathogenic bacteria cause serious chronic infections due to high tolerance to antibiotics and host immune systems compared to their planktonic compartments. Biofilm formation is known to be influenced by many properties of substrate materials, such as surface chemistry, hydrophobicity, roughness, topography, and charge. However, few studies have been conducted to investigate the effects of substrate stiffness. In this study, Escherichia coli RP437 and Pseudomonas aeruginosa PAO1 were used as model strains to investigate the early stage biofilm formation on poly(dimethylsil-oxane) (PDMS) with varying stiffness of 0.1 MPa to 2.6 MPa, which were prepared by controlling the degree of crosslinking. An inverse correlation between cell adhesion and substrate stiffness was observed for both E. coli and P. aeruginosa. Interestingly, it was found that the cells attached on relatively stiff substrates were significantly shorter than those on relatively soft substrates, and the distribution of cell length was narrower on stiff substrates. In addition to the difference in size, the cells on stiff substrates were also found to be less susceptible to antimicrobials, such as ofloxacin, ampicillin, tobramycin and lysozyme, than the cells attached on soft substrates. The cell tracking results revealed that the E. coli cells on stiff surfaces were more mobile than those on soft surfaces, suggesting that the cells attached on soft surfaces may enter biofilm stage faster. Consistently, the intracellular level of c-di-GMP (an important signal for biofilm formation) in the cells on soft surfaces was higher than that of cells on stiff surfaces. Comparison of the wild-type strains and isogenic mutants revealed that the motB mutant of E. coli RP437 has defects in response to the stiffness of PDMS, which was rescued by complementation of the motB gene. Additionally, the cell tracking results indicate that the mutation of motB rendered the cells much less mobile compared to wild type E. coli RP437 strains, and the decrease in the velocity of motility is higher on stiff surfaces than on soft surfaces. Those results suggest that motB may play a role in mechanosensing of material stiffness by E. coli. Similarly, mutation of oprF in P. aeruginosa also caused major defects in response to PDMS stiffness and abolished the difference in adhesion, growth, morphology and antibiotic susceptibility of attached cells between soft and stiff PDMS surfaces. These defects were rescued by genetic complementation of oprF, suggesting that oprF is involved in mechnosensing of P. aeruginosa. In summary, the findings from this study indicate that material stiffness has potent effects on bacterial adhesion and the physiology of attached cells. To our best knowledge, this is the first study on the effects of material stiffness of silicon-based polymers on biofilm formation, and the first report of the effects of material stiffness on the physiology of attached cells. These results are helpful for designing better anti-fouling materials

Actual Measurement and Analysis on Microbial Contamination in Central Air Conditioning System at a Venue in Dalian, China

AbstractActual measurement and analysis were carried out on microbial contamination in central air conditioning system at a venue in Dalian. By studying the microbial contamination in two air handling units with different thermal environments, we found that the fungi and bacteria were common existing on the surface of filter, and the trend of cell density distribution was center > against the wall > corner; The microbial pollution associated in the dust and floating in the air was extremely serious. By comparing the two units, we observed that fungus concentration: Unit A > Unit B, and bacteria concentration: Unit A < Unit B,. And the candida spp. accounted for 80 percent of the sample in Unit A; while in Unit B the cladosporium spp. occupied up to 50%. At the end of the paper, according to the results of measurement and analysis, the methods of controlling microbial contamination in HVAC system have been proposed

Cyclic-di-GMP and oprF Are Involved in the Response of Pseudomonas aeruginosa to Substrate Material Stiffness during Attachment on Polydimethylsiloxane (PDMS)

Recently, we reported that the stiffness of poly(dimethylsiloxane) (PDMS) affects the attachment of Pseudomonas aeruginosa, and the morphology and antibiotic susceptibility of attached cells. To further understand how P. aeruginosa responses to material stiffness during attachment, the wild-type P. aeruginosa PAO1 and several isogenic mutants were characterized for their attachment on soft and stiff PDMS. Compared to the wild-type strain, mutation of the oprF gene abolished the differences in attachment, growth, and size of attached cells between soft and stiff PDMS surfaces. These defects were rescued by genetic complementation of oprF. We also found that the wild-type P. aeruginosa PAO1 cells attached on soft (40:1) PDMS have higher level of intracellular cyclic dimeric guanosine monophosphate (c-di-GMP), a key regulator of biofilm formation, compared to those on stiff (5:1) PDMS surfaces. Consistently, the mutants of fleQ and wspF, which have similar high-level c-di-GMP as the oprF mutant, exhibited defects in response to PDMS stiffness during attachment. Collectively, the results from this study suggest that P. aeruginosa can sense the stiffness of substrate material during attachment and respond to such mechanical cues by adjusting c-di-GMP level and thus the following biofilm formation. Further understanding of the related genes and pathways will provide new insights into bacterial mechanosensing and help develop better antifouling materials

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis.

Bacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S rRNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary for analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which is time-consuming, expensive, and often biased based on the types of species present. Direct PCR method is a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, including both Gram-positive and Gram-negative species, ZymoBIOMICS microbial community standards, and groundwater. By comparing direct PCR methods with DNeasy Blood and Tissue Kits for microbial isolates and DNeasy PowerSoil Kits for microbial communities, we found that a specific variant of the direct PCR method exhibits an overall efficiency comparable to that of the conventional DNeasy PowerSoil protocol in the circumstances we tested. We also found that the method showed higher efficiency for extracting gDNA from the Gram-negative strains compared to DNeasy Blood and Tissue protocol. This direct PCR method is 1,600 times less expensive ($0.34 for 96 samples) and 10 times simpler (15 min hands-on time for 96 samples) than the DNeasy PowerSoil protocol. The direct PCR method can also be fully automated and is compatible with small-volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis. IMPORTANCE Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples. A direct PCR method could potentially solve these problems. In this study, we developed a direct PCR method which exhibits similar efficiency as the widely used method, the DNeasy PowerSoil protocol, while being 1,600 times less expensive and 10 times faster to execute. This simple, cost-effective, and automation-friendly direct-PCR-based 16S rRNA sequencing method allows us to study the dynamics, microbial interaction, and assembly of various microbial communities in a high-throughput fashion

How Bacteria Respond to Material Stiffness during Attachment: A Role of <i>Escherichia coli</i> Flagellar Motility

Material stiffness has been shown to have potent effects on bacterial attachment and biofilm formation, but the mechanism is still unknown. In this study, response to material stiffness by <i>Escherichia coli</i> during attachment was investigated with biofilm assays and cell tracking using the Automated Contour-base Tracking for in Vitro Environments (ACT<i>IV</i>E) computational algorithm. By comparing the movement of <i>E. coli</i> cells attached on poly­(dimethylsiloxane) (PDMS) surfaces of different Young’s moduli (0.1 and 2.6 MPa, prepared by controlling the degree of cross-linking) using ACT<i>IV</i>E, attached cells on stiff surfaces were found more motile during early stage biofilm formation than those on soft surfaces. To investigate if motility is important to bacterial response to material stiffness, we compared <i>E. coli</i> RP437 and its isogenic mutants of flagellar motor (<i>motB</i>) and synthesis of flagella (<i>fliC</i>) and type I fimbriae (<i>fimA</i>) for attachment on 0.1 and 2.6 MPa PDMS surfaces. The <i>motB</i> mutant exhibited defects in response to PDMS stiffness (based on cell counting and tracking with ACT<i>IV</i>E), which was recovered by complementing the <i>motB</i> gene. Unlike <i>motB</i> results, mutants of <i>fliC</i> and <i>fimA</i> did not show significant defects on both face-up and face-down surfaces. Collectively, these findings suggest that <i>E. coli</i> cells can actively respond to material stiffness during biofilm formation, and <i>motB</i> is involved in this response

NRF2 activation ameliorates blood–brain barrier injury after cerebral ischemic stroke by regulating ferroptosis and inflammation

Abstract Arterial occlusion-induced ischemic stroke (IS) is a highly frequent stroke subtype. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that modulates antioxidant genes. Its role in IS is still unelucidated. The current study focused on constructing a transient middle cerebral artery occlusion (tMCAO) model for investigating the NRF2-related mechanism underlying cerebral ischemia/reperfusion (I/R) injury. Each male C57BL/6 mouse was injected with/with no specific NRF2 activator post-tMCAO. Changes in blood–brain barrier (BBB)-associated molecule levels were analyzed using western-blotting, PCR, immunohistochemistry, and immunofluorescence analysis. NRF2 levels within cerebral I/R model decreased at 24-h post-ischemia. NRF2 activation improved brain edema, infarct volume, and neurological deficits after MCAO/R. Similarly, sulforaphane (SFN) prevented the down-regulated tight junction proteins occludin and zonula occludens 1 (ZO-1) and reduced the up-regulated aquaporin 4 (AQP4) and matrix metalloproteinase 9 (MMP9) after tMCAO. Collectively, NRF2 exerted a critical effect on preserving BBB integrity modulating ferroptosis and inflammation. Because NRF2 is related to BBB injury regulation following cerebral I/R, this provides a potential therapeutic target and throws light on the underlying mechanism for clinically treating IS

High-quality quantum process tomography of time-bin qubit’s transmission over a metropolitan fiber network and its application

We employ quantum state and process tomography with time-bin qubits to benchmark a city-wide metropolitan quantum communication system. Over this network, we implement real-time feedback control systems for stabilizing the phase of the time-bin qubits, and obtain a 99.3% quantum process fidelity to the ideal channel, indicating the high quality of the whole quantum communication system. This allows us to implement field trial of high performance quantum key distribution using coherent one way protocol with average quantum bit error rate and visibility of 0.25% and 99.2% during 12 hours over 61 km. Our results pave the way for the high-performance quantum network with metropolitan fibers.Comment: 6 pages, 4 figure

Quantifying changes in shoulder orientation between the prone and supine positions from magnetic resonance imaging

Background: Predicting breast tissue motion using biomechanical models can provide navigational guidance during breast cancer treatment procedures. These models typically do not account for changes in posture between procedures. Difference in shoulder position can alter the shape of the pectoral muscles and breast. A greater understanding of the differences in the shoulder orientation between prone and supine could improve the accuracy of breast biomechanical models. Methods: 19 landmarks were placed on the sternum, clavicle, scapula, and humerus of the shoulder girdle in prone and supine breast MRIs (N = 10). These landmarks were used in an optimization framework to fit subject-specific skeletal models and compare joint angles of the shoulder girdle between these positions. Findings: The mean Euclidean distance between joint locations from the fitted skeletal model and the manually identified joint locations was 15.7 mm ± 2.7 mm. Significant differences were observed between prone and supine. Compared to supine position, the shoulder girdle in the prone position had the lateral end of the clavicle in more anterior translation (i.e., scapula more protracted) (P \u3c 0.05), the scapula in more protraction (P \u3c 0.01), the scapula in more upward rotation (associated with humerus elevation) (P \u3c 0.05); and the humerus more elevated (P \u3c 0.05) for both the left and right sides. Interpretation: Shoulder girdle orientation was found to be different between prone and supine. These differences would affect the shape of multiple pectoral muscles, which would affect breast shape and the accuracy of biomechanical models